基于转录组测序的脓毒症休克转录因子挖掘及验证

Mining and validation of septic shock transcription factors based on transcriptome sequencing

目的通过mRNA高通量测序探讨脓毒症患者转为脓毒症休克的分子机制。方法选择2019年6月—2021年6月在新疆医科大学第四附属医院急诊科住院诊断为脓毒症患者70例作为研究对象,根据随访患者病情及预后,分为脓毒症休克组、脓毒症非休克组,每组35例。分析两组一般情况、感染指标方面的差异,通过Illumina测序平台,每组选取10例患者外周全血样本进行mRNA转录组测序,分析筛选差异表达基因,并对差异表达基因进行基因本体(gene ontology,GO)及基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)数据库富集分析,经蛋白质网络互作分析,筛选关键基因,进一步扩大样本至测序例数的两倍,并采取实时荧光定量检测(qPCR)法进行验证。结果两组一般情况中年龄、体重比较,差异无统计学意义(P>0.05);两组白细胞介素-6、降钙素原、C-反应蛋白比较,差异具有统计学意义(P<0.05);通过筛选共获得28543个差异基因(P<0.05),其中下调基因1126个,上调基因1053个;差异基因分析结果显示,排名前十的STAT1、MYD88及JUN2均为Toll样受体信号通路(toll like receptors,TLRs)相关基因;GO及KEGG富集分析发现,NOD样受体信号通路、TLRs信号通路、RIG-1样受体信号通路等与脓毒症的发生、发展相关;对TLRs信号通路进行蛋白质网络互作分析,筛选出该通路关键基因,对MYD88、JUN、CXCL10、STAT14个基因进行qPCR验证,结果与转录组分析一致。结论脓毒症患者的炎症反应可能与人体天然免疫系统的TLRs通路异常有关,MYD88、JUN、CXCL10、STAT1是导致脓毒症发展为脓毒症休克的关键基因,可为进一步研究脓毒症休克的发病机制奠定基础。

Objective To explore the molecular mechanism of septic shock in patients with sepsis by high-throughput mRNA sequencing.Methods 70 patients with sepsis hospitalized in the emergency department of the hospital from June 2019 to June 2021 were selected as the research object.According to the following condition and prognosis,the patients were divided into the sepsis shock group and the sepsis non shock group,with 35 cases in each group.The differences in general conditions and infection indexes between the two groups were analyzed.Through Illumina sequencing platform,10 patients in each group were selected for peripheral whole blood samples of mRNA transcriptome sequencing,and data analysis was to find differential genes.Gene Ontology(go)and genome Encyclopedia(KEGG)of differentially expressed genes were analyzed database enrichment analysis and protein network interaction analysis in order to screening of the key genes and to further expanding the sample for twice the number of sequencing cases of qPCR verification.Results There was no significant difference in general conditions,age and weight between the two groups(P>0.05);The difference of interleukin-6,procalcitonin and C-reactive protein between the two groups was statistically significant(P<0.05),which was consistent with the severity of the patient’s condition;A total of 28543 differential genes were obtained(P<0.05),including 1126 down-regulated genes and 1053 up-regulated genes.Differential gene analysis showed STAT1,MyD88 and jun2 were among the top ten and were related to toll like receptors(TLRs).After enrichment and analysis of go and KEGG databases,the founding was nod like receptor signal pathway,TLRs signal pathway and Rig-1 like receptor signal pathway were related to the occurrence and development of sepsis.The key genes of TLRs signaling pathway were screened by the protein network interaction analysis.The four genes MyD88,Jun,CXCL10 and STAT1 were verified by qPCR.The results were consistent with transcriptome analysis.Conclusion The inflammatory response of the patients with sepsis may be related to the abnormal TLRs pathway of human natural immune system.The key genes lead to the development of sepsis into septic shock are obtained,which lays a foundation for the further study of pathogenesis of septic shock.